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Concurrently with the above, we tested the growth of our organisms under different circumstances and on different media to see what ramifications the effects would have for our proposed co-culture.

LW06 on Maltose

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  • Figure 1: Maltose test plate 1 box plot. Plate 1 has serious anomalies in the blanks and controls when compared to plates 2 and 3. RV on y axis is the regression values. Results from this experiment where discounted due to problems with the control and blank samples.

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  • Figure 2: Maltose test plate 2 box plot. Missing data points in the blanks and controls are due to OD data not being high enough to be logged by R studio software. RV on y axis is the regression values.
    
    
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  • Figure 3: Maltose test plate 3 box plot. RV on y axis is the regression values.
    
    
    

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  • Figure 4: Maltose test plate 1 column graph, mean growth rates on maltose gradient in 96 well plates. Media maltose (1-8) represents columns 5–12 (TP.Maltose) wells. Given previously described issues with controls data from this experiment is not to be used for analysis.
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  • Figure 5: Maltose test plate 2 column graphs, mean growth rates on maltose gradient in 96 well plates. Media maltose (1-8) represents columns 5–12 (TP.Maltose) wells.

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  • Figure 6: Maltose plate 3 column graph, mean growth rates on maltose gradient in 96 well plates. Media maltose (1-8) represents columns 5–12 (TP.Maltose) wells.

Results: Consistency is shown for high growth in column 5 (1g/L) but remains more or less level throughout the increasing maltose concentrations. Such consistency indicates LW06 is somewhat poor at maltose metabolism but is able to survive on it. Mean growth rates are small, though this is potentially explained by a highly limiting culture volume. However, data from the plate reader indicates clear exponential and plateau events during growth. In conclusion, growth under maltose conditions is possible. Conditioning for maltose consumption may be required to improve final culturing with C. reinhardtii when it is exporting maltose, in future co-cultures. The problems with the first experimental run, contamination in the blanks and controls, were most likely due to condensation on the interior lid or displacement of culture material in the controls and blank sections of the plate during agitation. As a result of this, data from this experiment is to be discounted for analysis and should be considered when proceeding with experiments in the future as to avoid issues of contamination.

LW06 on Home Blend Medium

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  • Figure 1: Home blend (Hblend) test plate 1. Regression values (y axis) show that TP-Maltose performs worse than TP-Glucose. Hblend-Glucose greatly out performs Hblend-Maltose, but is comparable to TP-Maltose.

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  • Figure 2: Hblend test plate 1. Mean growth rates here show that the growth rate for Hblend-Glucose is much greater than TP or Hblend-Maltose. TP-Maltose outperforms our Home blend recipe.
• A T-test was conducted for TP.Maltose and Hblend.Maltose. T-test (Degrees of Freedom = 48, p = 8.82 x 10^-10). This indicates significant difference between media types.
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  • Figure 3: Hblend test plate 2. Regression values (y axis), as in figure 1, show that TP and Hblend-Maltose are out performed by Hblend-Glucose.

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  • Figure 4: Hblend test plate 2. Mean growth rates here show Hblend-Glucose out performs TP or Hblend-Maltose with no difference between the latter pair in terms of growth.
• A T-test was conducted for the second plate. T-test (D.o.F. = 48, p = 0.058255). For the second plate, no statistical significance between media types was indicated.

Results: Overall, Hblend performs unremarkably when compared to TP media. Despite having much higher micronutrients, Hblend does not perform as well as we hypothesised. Some data collected indicates that a longer run time may have had an effect, but it is unlikely that this would lead to major improvements over the TP media. The problem seems to be the uptake or metabolism of maltose. Conditioning our organisms to grow on maltose may improve growth rates in a co-culture. However, such a limitation may work to the advantage of the co-culture. A key problem to consider was the greatly divergent growth rates between C. reinhardtii and the LW06 E. coli strain. This reduced growth with maltose as the carbon source may provide a sufficient rate limiter on the growth of LW06 to avoid complete culture saturation with the E. coli in future co-cultures.

C. reinhardtii Ethanol Tolerance

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  • Figure 1: Cell counts for C. reinhardtii via multiple well plate readers. Readings taken ~24hours manually.

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  • Figure 2: Cell counts for C. reinhardtii via multiple well plate readers. Readings taken ~24hours manually.

Results: C. reinhardtii is able to tolerate an increase of ethanol to at the most 3.2% (as a portion of media). Given rates of increase it seems that it could tolerate much higher concentrations. This is encouraging, as ethanol resistance of the C. reinhardtii would likely be sufficient to survive upper limits of the LW06 strain’s ethanol production. Interestingly, C. reinhardtii experiences better growth at higher concentrations of ethanol, there is not a clear reason this is the case. Addition of ethanol to the media may have made growth conditions more amenable to C. reinhardtii. There is some chance that the ethanol may have evaporated which, while possible, is unlikely due to the sealing of the plate. Overall, it seems that C. reinhardtii would be able to survive the proposed amount of ethanol that could be produced by the LW06.

There's a large growth disparity between the two organisms (source???)(source???), so we also performed an experiment to check if LW06 would vastly outperform C. reinhardtii on all types of media.

Growth in a Co-culture

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  • Figure 1: Cell counts for LW06 in each culture via spread plating at 10⁴ dilutions.

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  • Figure 2: Cell counts for C. reinhardtii via manual haemocytometer microscope cell counting at a 10⁴ dilution.
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  • Figure 3: Overlaid cell counts for the co-culture. LW06 represented by blue points and C. reinhardtii by orange.

Results: Overall LW06 outperforms C. reinhardtii in all but one of the cultures (Tube 4). It is encouraging to see LW06 perform well on TP.Maltose and TAP.Maltose, although the latter may have been due to the presence of a second carbon source in the form of acetate. It is odd that in every one of the experiments tube that were inoculated with 100 μl of 0.5-1.0 OD LW06 that the cell counts are considerably lower. It seems that the initial starting cell density may have inhibited growth but it is not clear why this effect would continue when dispersed in the co-culture. Alternatively, an unknown error may have occurred at some point during inoculation. From the results it is clear that, if it was some unknown or unnoticed error, it was made consistently in every one of the co-cultures with 100 μL inoculation. No relevant conclusions can be drawn from this. However, considerations such as the growth limiting factor of the TP.Maltose may work in our interest as to avoid culture saturation with LW06.

An obvious error, in hindsight, is the issue of the 10 μL inoculation. It is likely that a much higher volume added to the culture would have improved the results. This would have provided a high enough cell density, should growth have not be great enough during co-culturing with the modified C. reinhardtii in TP media. Overall co-cultures with these two organisms are possible, but higher OD/volume of inoculant may be needed.

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